Dengue (DV) disease is a major and spreading global health problem. Transmission is now endemic in at least one U.S. state; hundreds of veterans were affected by a DV outbreak in 2007-10. Most severe DV disease is mediated by antibody-mediated enhancement of infection (ADE) inwhich antibodies from a prior infection with another DV serotype or maternal-infant transmission facilitate viral entry and replication. A 7.5- fold increased risk of severe DV disease is seen in DV-nave recipients of the YF-vectored DV vaccine (CYD), indicating this vaccine is inducing ADE in those subjects. As neither vaccination nor repeated infection induce durable high titer neutralizing antibodies in sufficient numbers of people at risk, it is vital to determine how CMI blocks ADE-mediated DV replication in primary host cells so these responses can be targeted in vaccines. The macrophage is both the primary DV replication-permissive cell and a key contributor to the exaggerated inflammatory response seen in severe DV. Blocking DV replication in macrophages will both lower viremia and inflammation. It is therefore critical to identify people with CMI that can mediate cross-serotypic control of ADE-mediated DV replication in autologous macrophages. These people can then be further studied to identify cellular mechanisms/effector molecules which mediate control of ADE-enhanced DV replication. We hypothesize that a safe protective DV vaccine must induce effective durable CMI which blocks ADE-enhanced DV replication in the primary host cell.A Key Gap in knowledge is the CMI cells and phenotypes which inhibit DV replication in primary human cells are inadequately defined. We have optimized an ex vivo protection assay which measures DV-specific memory CMI inhibition of ADE-enhanced DV replication. Using samples collected from a TDV dengue vaccine trial at Saint Louis University, we have shown that TDV vaccination of flavivirus-naives can induce cross-serotype CMI capable of inhibiting ADE-mediated DV replication in macrophages. Using this assay, we will: 1.Determine whether TDV vaccination induces durable cross-protective CMI in DV naives which inhibits ADE-enhanced DV replication. 2.Determine if CMI after TDV vaccination is comparable to CMI after sequential natural infections. 3.Identify key CMI functions associated with control of ADE-enhanced DV replication in MDMs. 4.Determine the durability of DV replication-inhibitory memory CMI after TDV vaccination of DV-naives. 5.Determine the prevalence of prior DV infection in a representative cohort of DV-exposed veterans and measure the prevalence of cross-serotype protective CMI years after exposure. Our data will answer the following questions: 1. Does TDV vaccination induce durable CMI able to control ADE-enhanced DV replication in primary cells in flavivirus-naives? DV-naives cannot receive the CYD vaccine so it is vital to identify a safe vaccine for this population. 2. Since repeated DV infections induce protection from severe disease, CMI from these people can be used as an approximation of protective immunity. Does TDV- induced CMI approximate CMI after repeated infections? 3. What key cells/effector molecules are vital in inhibition of DV replication in macrophages? 4. What is the prevalence of DV seropositivity in veterans and the durability of CMI? Our data will inform vaccine development by identifying key protective cell types and effector functions to be targeted by vaccines. If we are going to control the DV pandemic, we need to identify what cells/effector functions of human cellular immunity can control ADE-mediated DV replication in the primary host cell. This application can answer those questions.